Process for the preparation of epoxides of means of microorganisms

ABSTRACT

2,3-Epoxypropyl ethers valuable as intermediates for preparing medicaments and the like are prepared from the corresponding allyl ethers by means of epoxide-producing microorganisms belonging to Nocardia, Brevibacterium, Corynebacterium, Pseudomonas, Rhodococcus, Arthrobacter or Micrococcus.

This application is a continuation of application Ser. No. 07/700,326filed May 9, 1991, now abandoned, which is a continuation of applicationSer. No. 07/268,648 filed Nov. 8, 1988, now abandoned, which is acontinuation of application Ser. No. 06/737,966 filed May 28, 1985, nowabandoned.

FIELD OF THE INVENTION

The present invention relates to a process for the preparation ofepoxides by means of microorganisms, more particularly, a process forthe preparation of 2,3-epoxypropyl ethers from the corresponding allylethers by means of microorganisms.

BACKGROUND OF THE INVENTION

2,3-Epoxypropyl phenyl ethers, a sort of 2,3-epoxypropyl ethers, areimportant as starting materials for medicaments, particularly, asintermediates for syntheses of β-blockers, since they can be convertedinto aryloxypropanolamines by ring cleavage using amines, as shown bythe following reaction formula: ##STR1##

Also, 2,3-epoxypropyl naphthyl ethers are important, like the2,3-epoxypropyl phenyl ethers, as intermediates for syntheses ofβ-blockers. Further, various valuable compounds were synthesized from2,3-epoxypropyl benzyl ethers via γ-butyrolactones.

The 2,3-epoxypropyl ethers obtainable by the process of the presentinvention are important as intermediates for the preparation ofmedicaments, agricultural chemicals and the like, since they areoptically active and so various optically active derivatives can besynthesized starting from them.

Hitherto, as processes for preparing epoxides from the correspondingolefins were known chemical oxidation processes using a peroxide such ashydrogen peroxide or percarboxylic acids as an oxidizing agent, andbiochemical oxidation processes using microorganisms. According to thebiochemical processes using microorganisms, epoxides were prepared bytreating the corresponding linear olefins or the correspondingalkenylbenzenes such as styrene, allylbenzene or the like, withmicroorganisms belonging to Nocardia, Pseudomonas, Brevibacterium,Corynebacterium, Mycobacterium, Arthrobacter, Acinetobacter,Alcaligenes, Methylobacterium, Methylococcus, Methylocinus, or the like.

In the epoxidation by means of microorganisms, however, the sorts ofolefins which can be epoxidized were limited depending on the sort ofthe microorganism used. For example, it was reported that Pseudomonasoleovorans did not epoxidize propylene, 1-butene, 2-octene,cis-5-decene, cyclohexene and styrene [S. W. May, R. D. Schwartz, B. J.Abbott and O. S. Zaborsky, Biochim. Biophys. Acta., 403, 245-255(1975)], although α-olefins of C₆ to C₁₂ [B. J. Abbott and C. T. Hou,Appl. Microbiol., 26, 86-91 (1973)], α,ω-dienes [S. W. May, R. D.Schwartz, B. J. Abbott and O. S. Zaborsky, Biochim. Biophys. Acta.,403,245-255 (1975)] and allylbenzene [M-J de Smet, J. Kingma, H. Wynbergand B. Witholt, Enzyme Microb. Technol., 5, 352-360 (1983)] wereepoxidized.

On the other hand, Nocardia corallina epoxidized α-olefins of C₃ to C₁₈(Japanese Patent Publication No. 40/81) and also inner olefins such as2-octene, 3-octene and the like (Japanese Patent Laid-Open No. 141791/83).

Thus, the sorts of olefins which can be epoxidized differ depending onthe sort of the microorganism used, and accordingly it is necessary toexamine individual olefins and individual microorganisms. As for theoxygen-containing unsaturated compounds such as allyl ethers, amongcompounds having a C--C double bond, any process for preparing epoxidesfrom them by means of microorganisms is not known as yet.

Moreover, as for the preparation of optically active epoxides by meansof microorganisms, any process for preparing optically active epoxidesfrom oxygen-containing unsaturated compounds such as allyl ethers bymeans microorganisms is not known as yet, although it is known thatoptically active epoxides are formed in the preparation of epoxides fromlinear olefins by microorganisms belonging to Corynebacterium orPseudomonas and also in the preparation of epoxides from allylbenzene bymicroorganisms belonging to Pseudomonas.

Now, after making search for microorganisms capable of producingepoxides from allyl ethers, it has been found that epoxide-producingmicroorganisms belonging to Arthrobacter, Brevibacterium,Corynebacterium, Micrococcus, Nocardia, Pseudomonas or Rhodococcusproduce epoxides from the corresponding allyl ethers and that theepoxides produced are optically active. Thus, the present invention hasbeen accomplished.

That is, the object of the present invention resides in providing anovel process for the preparation of 2,3-epoxypropyl ethers valuable asintermediates for preparing medicaments and the like, by means ofepoxide-producing microorganisms belonging to Arthrobacter,Brevibacterium, Corynebacterium, Micrococcus, Nocardia, Pseudomonas orRhodococcus.

SUMMARY OF THE INVENTION

A characteristic feature of the present invention resides in producing2,3-epoxypropyl ethers by treating the corresponding allyl ethers of thegeneral formula X--O--CH₂ --CH═CH₂ (I), as defined hereinafter, withmicroorganisms having an ability of producing epoxides which areselected from the group of microorganisms belonging to Arthrobacter,Brevibacterium, Corynebacterium, Micrococcus, Nocardia, Pseudomonas orRhodococcus under an aerobic condition, and isolating the epoxidesproduced.

A further characteristic feature of the present invention resides inproducing the epoxides more advantageously by effecting the treatmentwith the above-mentioned microorganisms in the presence of awater-insoluble organic solvent.

Examples of the starting materials and the epoxides produced therefromaccording to the present invention are shown by the following schemes:##STR2##

DESCRIPTION OF THE PREFERRED EMBODIMENT

As examples of the microorganisms belonging to Arthrobacter,Brevibacterium, Corynebacterium, Microcococcus, Nocardia, Pseudomonas orRhodococcus, which are used in the present invention, there can bementioned those strains as listed in the following Table 1. Thesestrains are easily available, being deposited at American Type CultureCollection under the respective ATCC numbers.

                  TABLE 1                                                         ______________________________________                                         1. Arthrobacter roseoparaffinus                                                                  ATCC 15584                                                 2. Arthrobacter petroleophagus                                                                   ATCC 21494                                                 3. Arthrobacter rubellus                                                                         ATCC 21495                                                 4. Arthrobacter sp.                                                                              ATCC 27778                                                 5. Brevibacterium butanicum                                                                      ATCC 21196                                                 6. Corynebacterium fujiokense                                                                    ATCC 21496                                                 7. Rhodococcus sp. ATCC 15108                                                 8. Micrococcus paraffinolyticus                                                                  ATCC 15589                                                 9. Nocardia corallina                                                                            ATCC 31338                                                10. Rhodoccus rhodochrous                                                                         ATCC 21197                                                11. Rhodoccus rhodochrous                                                                         ATCC 21198                                                12. Pseudomonas oleovorans                                                                        ATCC 29347                                                13. Rhodococcus rhodochrous                                                                       ATCC 29675                                                14. Rhodococcus rhodochrous                                                                       ATCC 29670                                                15. Rhodococcus rhodochrous                                                                       ATCC 29672                                                16. Rhodococcus sp. ATCC 29673                                                17. Rhodococcus sp. ATCC 29674                                                ______________________________________                                    

The allyl ethers which are used in the present invention as substratesfor producing epoxides by treating with the above microorganisms arethose of the general formula (I)

    X--O--CH.sub.2 --CH═CH.sub.2                           (I)

wherein X represents a radical selected from the group consisting of

(a) phenyl and substituted phenyl radicals ##STR3## in which R₁ denoteseither one, or two or more, selected from the group consisting ofhydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,t-butyl, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy,t-butoxy, halogeno, cyano, cyclopentyl, allyl, allyloxy, methoxyethyland benzyloxy, and n is an integer of 1 to 3, said R₁ 's being the sameor different from one another when n is an integer of 2 to 3,

(b) α- and β-naphthyl radicals and their substituted radicals ##STR4##in which R₂ denotes either one, or two or more, selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and iso-propyl, and n isan integer of 1 to 4, said R₂ 's being the same or different from oneanother when n is an integer of 2 to 4, and

(c) benzyl radical ##STR5##

More particularly, allyl phenyl ether, α-naphthyl allyl ether,2-methylphenyl allyl ether, 4-n-butylphenyl allyl ether,2,5-dimethylphenyl allyl ether, 2,3-dimethylphenol allyl ether,2,6-di-t-butyl-4-methylphenyl allyl ether, 2-allylphenyl allyl ether,2-cyanophenyl allyl ether, 2-cyclopentylphenyl allyl ether, 2-methoxyphenyl allyl ether, 2-chlorophenyl allyl ether, 2-allyloxyphenylallyl ether, 4-β-methoxyethylphenyt allyl ether, 4-butoxyphenyl allylether, 3-methylphenyl allyl ether, 2-chloro-5-methylphenyl allyl ether,benzyl allyl ether and the like can be mentioned as examples of theallyl ethers.

According to the present invention, these allyl ethers are used assubstrates, individually or as a mixture of two or more of them.

The allyl group of these allyl ethers is epoxidized by themicroorganisms. Among the above-mentioned allyl ethers, allylphenylallyl ether is epoxidized only at the allyl group bonded by etherlinkage, and allyloxyphenyl allyl ether is epoxidized only at one allylgroup. The epoxides produced according to the present invention areoptically active.

To produce epoxides from the allyl ethers by the microorganismsbelonging to Arthrobacter, Brevibacterium, Corynebacterium, Micrococcus,Nocardia, Pseudomonas and Rhodococcus, according to the presentinvention, (a) a method of incubating the allyl ethers under an aerobiccondition with the precultivated cells of the microorganisms or (b) amethod of cultivating the microorganisms under an aerobic condition in aculture medium containing the allyl ethers may be applied.

In the method (a) of incubating the allyl ethers with the precultivatedcells, the microorganism precultured under an aerobic condition in amedium consisting of assimilable carbon sources such as glucose sucrose,molasses, starchhydrolysate, propane, butane, octane, dodecane,tetradecane, ethylene, propyl end, 1-butene, 1,3-butadiene, acetic acid,ethanol, etc., or sources such as ammonium chloride, ammonium sulfate,ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, aminoacidsand other assimilable organic nitrogen compounds, inorganic salts suchas potassium phosphate, sodium phosphate, magnesium sulfate, manganesesulfate, ferrous sulfate, ferric chloride, calcium chloride, manganesechloride, etc., salts of boron, cooper, zinc, etc., i.e., the so-called"trace elements", and if necessary, those substances which stimulate thegrowth of microorganism, such as vitamins, yeast extract, corn steedliquor, etc. To the cultured solution thus obtained, or to a suspensionof the cells harvested therefrom or immobilized cells preparedtherefrom, are added the ally ethers and, if necessary, these organicsolvents as described hereinafter, and then an oxygen-containing gassuch as air, oxygen gas, oxygen-enriched gas, etc. is provided to causethe reaction.

The reaction is performed at a pH of 5 to 9 and a temperature of 20° to50° C. for 1/2 to 6 days, as suitably prescribed depending on themicroorganisms and allyl ethers used. Although the reaction is performedusually under normal pressure, it is also possible to improve theepoxide-productivity by carrying out the reaction under elevatedpressure. In addition, it is possible to maintain or elevate the cellconcentration and the epoxide-productivity of cells by suitably addingthe carbon source, the nitrogen source and the other ingredients as usedin the cell-cultivation, to the reaction mixture in the course of thereaction.

The ratio of the allyl ethers to the aqueous liquor containing cellsused in the reaction is usually 0.1 to 50 vol/vol %, preferably, 0.5 to20 vol/vol %.

The reaction can be performed by either of a batchwise process or acontinuous process, or also by a semicontinuous process wherein theallyl ethers or other ingredients are supplemented continuously orintermittently in the course of the reaction.

The epoxides produced by the reaction are separated and purified byconventional methods such as phase separation, extraction, distillationand the like.

The method (b) is intended for producing epoxides by one step by addingthe allyl ethers and, if necessary, those organic solvents as describedhereinafter to the medium for the precultivation in the method (a). Asconditions for cultivation (such as pH, temperature, pressure, amount ofallyl ethers added, etc.), mode of cultivation and means of separationand purification of epoxides, the same reaction conditions, reactionmode and means for separation and purification as employed in the method(a) may be used.

As described above, the present invention encompasses or covers also anembodiment of performing the reaction of epoxidizing the allyl etherswith the microorganisms, in the presence of water-insoluble organicsolvents. Therefore, such embodiment is explained hereunder.

The water-insoluble organic solvents which may be present on carryingout the epoxidation of the allyl ethers by means of the microorganisms(hereinafter, referred to merely as organic solvents) are those organicsolvents selected from the group consisting of paraffins of C₉ to C₁₇,olefins of C₁₀ to C₁₈, halogenated paraffins of C₉ to C₁₆ andalkylbenzenes having one alkyl side chain of C₆ to C₁₅. These organicsolvents may be used individually or as a mixture of two or more ofthem.

Now, more detailed explanation is given as to the organic solvents.

Among paraffins of C₉ to C₁₇, normal paraffins are those contained inkerosene and gas oil fractions of petroleum in an amount of about 20 to25%. They are separated and recovered by means of zeolite (or molecularsieve) or the like, after hydrodesulfurization of the fraction havingboiling points of about 160° to 350° C. They are generally used asmaterials for soft-type detergents.

The above paraffins containing a number of carbon atoms toward the highend of the range have a higher epoxidation-accelerating effect and theof C₁₂ to C₁₆ paraffins are especially preferred. By the way, thosecontaining less than 9 carbon atoms do not show anyepoxidation-accelerating effect and, on the other hand, those containingmore than 17 carbon atoms are of no practical use because they show onlya lower epoxidation-accelerating effect and moreover they solidify atroom temperature.

Further, among the above paraffins, isoparaffins are those coexistingwith the normal paraffins in the above-mentioned fractions. Althoughthey can be separated from normal paraffins by rectification, it isconvenient in practice to use them in the form of mixtures with normalparaffins. They generally have side chains of short length, such asmethyl, ethyl and the like. Isoparaffins of C₁₂ to C₁₆ are especiallypreferable for the purpose of accelerating epoxidation.

The olefins of C₁₀ to C₁₈ may be lower polymers or oligomers ofpropylene or butylene. Those commercially available as reagents may alsobe applied. Generally, they are linear or lower branched monoolefins.

By the way, olefins containing less than 10 carbon atoms do not show anyepoxidation-accelerating effect and, on the other hand, those containingmore than 18 carbon atoms are of no practical use because they show onlya lower epoxidation-accelerating effect and moreover they are highlyviscous.

The halogenated paraffins of C₉ to C₁₆ as the organic solvents are thosechlorinated paraffins and brominated paraffins which include decylchloride, undecyl chloride, undecyl bromide, dodecyl bromide, tetradecylbromide, hexadecyl bromide, etc. Both the halogenated paraffinscontaining less than 9 carbon atoms and the halogenated paraffinscontaining more than 16 carbon atoms do not show anyepoxidation-accelerating effect.

The alkylbenzenes having one alkyl side chain of C₆ to C₁₅ are thoseusually utilized as intermediates for hard or soft detergents, whoseside chain is a linear or branched alkyl group. When the length of theside chain is outside the range of C₆ to C₁₅, anyepoxidation-accelerating effect is not recognized or theepoxidation-accelerating effect is too low to be used practically.

The ratio of the above-mentioned organic solvents to the cultured brothor the suspension of the cells may vary depending on the sorts of theorganic solvents used. However, it is usually 1 to 200 vol/vol %,preferably 5 to 100 vol/vol %.

The same reaction conditions, reaction mode and means for separation andpurification of the epoxides formed, as described above, may be appliedalso in the case of performing the reaction in the presence of theorganic solvents. The epoxide-productivity may be significantly elevatedby the presence of the organic solvents.

The epoxides obtained according to the present invention are opticallyactive and so they can be utilized advantageously, especially asstarting materials for synthesizing physiologically active substancessuch as medicaments.

Hereinafter, the present invention is further explained moreparticularly, by giving Examples. The invention, however, shall never belimited to these Examples

EXAMPLE 1 PREPARATION OF CELL SUSPENSION

As regards the 11 microorganisms listed in %he following Table 3 exceptPseudomonas oleovorans, a 100 ml of NBG medium (a liquid medium preparedby adding tap-water to 10 g of Lab-Lemco Powder from "Oxoid", 10 g ofbacteriological peptone, 10 g of glucose and 5 g of sodium chloride tomake the whole volume into 1 liter and adjusted the pH of the solutionat 7.5 with 1N-aqueous sodium hydroxide solution and then autoclaved at120° C. for 15 minutes) placed in a 500 ml shake flask was inoculatedwith 3 loopfuls of cells and cultivated at 30° C. for 48 hours underoscillation.

As for Pseudomonas oleovorans in Table 3, the microorganism wastransferred from a nutrient agar slant to a nutrient agar slantsupplemented with 1% of glucose and incubated at 30° C. for 24 hours. A100 ml of a minimal salt medium of the following Table 2 and 2 ml ofoctane placed in a 500 ml shake flask was inoculated with 2 loopfuls ofcells and cultivated at 30° C. for 16 hours under oscillation. Themedium was autoclaved at 120° C. for 20 minutes. Octane was sterilizedthrough a 0.2 μm membrane filter.

A cell suspension of each microorganism was prepared by washing theharvested cells once with 0.01M phosphate buffer (pH 7.5) and then oncewith the reaction medium as described hereinafter, and thereafterresuspending the cells in the same reaction medium. The concentration ofcells in the cell suspension was adjusted to be within the range of 3.5to 4.0 g/l in dry cell basis.

    ______________________________________                                        Reaction medium:                                                                              K.sub.2 HPO.sub.4                                                                           1.74   g                                                        MgSO.sub.4.H.sub.2 O                                                                        1.50   g                                                        FeSO.sub.4.7H.sub.2 O                                                                       0.05   g                                                        deionized water                                                                             1      l                                        ______________________________________                                    

The pH was adjusted at 8.0 with 2N-H₂ SO₄.

                  TABLE 2                                                         ______________________________________                                        Minimal salt medium                                                           ______________________________________                                        (NH.sub.4).sub.2 HPO.sub.4                                                                           10.0   g                                               K.sub.2 HPO.sub.4      5.0    g                                               Na.sub.2 SO.sub.4      0.5    g                                               CaCl.sub.2 (50 g/l)    1.0    ml                                              Salt "B"               10.0   ml                                              MgSO.sub.4.7H.sub.2 O  40.0   g                                               FeSO.sub.4.7H.sub.2 O  2.0    g                                               MnSO.sub.4.H.sub.2 O   1.6    g                                               NaCl                   2.0    g                                               distilled water        1      l                                               Trace metal solution   1.0    ml                                              H.sub.3 BO.sub.3       0.50   g                                               CuSO.sub.4.5H.sub.2 O  0.04   g                                               Na.sub.2 MoO.sub.4.H.sub.2 O                                                                         0.20   g                                               ZnSO.sub.2.7H.sub.2 O  8.00   g                                               CuCl.sub.2.6H.sub.2 O  0.20   g                                               distilled water        1      l                                               Distilled water        1      l                                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                                        Amounts                                                                       of the                                                                        epoxide                                                                       produced                                      Microorganism Used              (mg)                                          ______________________________________                                         1. Arthrobacter roseoparaffinus                                                                  ATCC 15584  0.8                                            2. Arthrobacter petroleophagus                                                                   ATCC 21494  0.06                                           3. Arthrobacter rubellus                                                                         ATCC 21495  0.1                                            4. Brevibacterium butanicum                                                                      ATCC 21196  0.7                                            5. Corynebacterium fujiokense                                                                    ATCC 21496  0.2                                            6. PseudoNonas oleovorans                                                                        ATCC 29347  0.02                                           7. Rhodococcus rhodochrous                                                                       ATCC 29670  0.1                                            8. Rhodococcus rhodochrous                                                                       ATCC 29672  0.1                                            9. Rhodococcus rhodochrous                                                                       ATCC 19675  0.05                                          10. Nocardia corallina                                                                            FERM-P-4094 10.3                                                              ATCC 31338                                                11. Rhodococcus rhodochrous                                                                       ATCC 21197  0.5                                           12. Nocardia paraffinica                                                                          ATCC 21198  0.2                                           ______________________________________                                    

REACTION AND ANALYSIS OF THE PRODUCT

In a 500 ml shake flask were placed 20 ml of the above cell suspensionand 1 ml of allyl phenyl ether. After incubation under oscillation at30° C. for 24 hours, the 2,3-epoxypropyl phenyl ether produced wasextracted with 40 ml of ether and its amount was determined by agaschromatograph equipped with a flame ionization detector and a glasscolumn 2 m long packed with 3% DEGS (diethylene glycol succinate) onUniport B 80-100 mesh.

RESULTS

The sorts of microorganisms used and the amounts of the epoxide producedby each microorganism are shown in Table 3.

EXAMPLE 2

Nocardia corallina ATCC 31338 (FERM-P-4094) was cultivated according tothe procedure described in Example 1 and its cell suspension wasprepared. The reaction was carried out in the same manner as describedin Example 1, by two methods, i.e., by a reaction method (a) wherein 1ml of each of the starting materials ethers as described in thefollowing Table 4 was added to 20 ml of the cell suspension, and areaction method (b) wherein 0.4 ml of each of the starting materialsethers and 2 ml of n-hexadecane were added to 20 ml of the cellsuspension.

The reaction was carried out either 24 or 72 hours. The sorts of theethers used as the starting material and the amounts of the epoxideproduced from each ether are shown in Table 4. The epoxides were assayedin the same manner as described in Example 1.

                  TABLE 4                                                         ______________________________________                                                            Amounts of the                                                         Reaction                                                                             epoxide produced (mg)                                     Ethers used as time     Reaction  Reaction                                    the starting material                                                                        (hours)  method (a)                                                                              method (b)                                  ______________________________________                                        2-methylphenyl allyl ether                                                                   24       19.0      36.0                                        3-methylphenyl allyl ether                                                                   24       12.7      25.0                                        4-methylphenyl allyl ether                                                                   24       23.3      47.3                                        2-allylphenyl allyl ether*                                                                   24       16.5      27.6                                                       72       20.4      64.9                                        2-allyloxyphenyl allyl                                                                       24       27.9      20.5                                        ether**                                                                       2-chloro-5-methylphenyl                                                                      72       0.1 or less                                                                             3.7                                         allyl ether                                                                   α-naphthyl allyl ether                                                                 72       0.1 or less                                                                             4.1                                         ______________________________________                                         *the product was 2allylphenoxymethyl oxirane                                  **the product was a monoepoxide                                          

EXAMPLE 3

Brevibacterium butanicum ATCC 21196 was cultivated according to theprocedure described in Example 1 and its cell suspension was prepared.The reaction was carried out in the same manner as described in Example1 by two methods, i.e., by a reaction method (a) wherein 1 ml of each ofthe starting materials ethers as described in the following Table 5 wasadded to 20 ml of the cell suspension, and a reaction method (b) wherein0.4 ml of each of the starting materials ethers and 2 ml of n-hexadecanewere added to 20 ml of the cell suspension.

The reaction was carried out for 24 hours. The sorts of the ethers usedas the starting material and the amounts of the epoxide produced fromeach ether are shown in Table 5. The epoxides were assayed in the samemanner as described in Example 1.

                  TABLE 5                                                         ______________________________________                                                          Amounts of the                                                                epoxide produced (mg)                                                          Reaction    Reaction                                       Ethers used as the starting material                                                             method (a)  method (b)                                     ______________________________________                                        2-methylphenyl allyl ether                                                                       1.5         3.2                                            3-methylphenyl allyl ether                                                                       1.0         2.0                                            4-methylphenyl allyl ether                                                                       1.8         3.5                                            2-allylphenyl allyl ether*                                                                       1.3         2.8                                            ______________________________________                                         *the product was 2allylphenoxymethyl oxirane                             

EXAMPLE 4

A cell suspension of Nocardia corallina ATCC 31338 was preparedaccording to the procedure described in Example 1. The cellconcentration was adjusted to 15 g/l in dry cell basis. 20 ml of thecell suspension, 1 ml of 2-allylphenyl allyl ether as the startingmaterial and 1 ml of each of the organic solvents described in thefollowing Table 6 were placed in a 500 ml shake flask, and the reactionwas carried out in the same manner as described in Example 1. After thereaction for 24 hours, the organic solvent layer was extracted with 40ml of ether and the amount of the 2-allylphenoxymethyloxirane producedwas determined according to the method described in Example 1. The sortsof the organic solvents used and the amounts of the epoxide produced areshown in Table 6.

                  TABLE 6                                                         ______________________________________                                                        Amounts of the                                                Organic solvents used                                                                         epoxide produced (mg)                                         ______________________________________                                        Paraffins                                                                     n-decane        110                                                           n-dodecane      125                                                           n-tridecane     140                                                           n-tetradecane   160                                                           n-hexadecane    130                                                           paraffin mixture*                                                                             150                                                           Olefins                                                                       1-dodecene       85                                                           1-hexadecene    115                                                           Halogenated paraffins                                                         1-chlorododecane                                                                              145                                                           1-chlorotetradecane                                                                           120                                                           Alkylbenzenes                                                                 dodecylbenzene  135                                                           tridecylbenzene 130                                                           ______________________________________                                         *a mixture of 95% by weight of each normal paraffin of C.sub.9 to C.sub.1     and 5% by weight of each isoparaffin of C.sub.9 to C.sub.16              

Next, for reference, the optical purities of the epoxides produced usingNocardia corallina ATCC 31338 are shown in the following Table 7.

By the way, the optical purity was determined according to the procedureof J. A. Dale, D. L. Dull and H. S. Mosher, J. Org. Chem., 34, 2453(1963). Namely, each epoxide was reduced with LiAlH₄ to thecorresponding 1-phenoxy-2-propanol, and then the latter was esterifiedwith (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride. By measuringNMR spectra of ¹⁹ F in the ester, the optical purity was calculated fromthe ratio of peak intensity of the diasteromers.

                  TABLE 7                                                         ______________________________________                                                           Optical                                                                       purity     Absolute                                        Epoxides           (% e.e.)   configuration                                   ______________________________________                                        phenoxymethyloxirane                                                                             67         S                                               (2-methylphenoxy)methyloxirane                                                                   73         S                                               (3-methylphenoxy)methyloxirane                                                                    5         S                                               (4-methylphenoxy)methyloxirane                                                                   79         S                                               (2-allylphenoxy)methyloxirane                                                                    53         S                                               (2-allyloxyphenoxy)methyloxirane                                                                 58         S                                               1-naphthoxymethyloxirane                                                                         71         R                                               ______________________________________                                    

EXAMPLE 5

A cell suspension of Brevibacterium butanicum ATCC 21196 was preparedaccording to the same procedure as described in Example 1. The cellconcentration was adjusted to 7 to 8 g/l in dry basis. 20 ml of the cellsuspension, 1 ml of 2-allylphenyl allyl ether as the starting materialand 1 ml of each of the organic solvents described in the followingTable 8 were placed in a 500 ml shake flask, and the reaction wascarried out in the same manner as described in Example 1. After thereaction for 24 hours, the organic layer was extracted with 40 ml ofether and the amount of 2-allylphenoxymethyloxirane produced wasdetermined according to the method described in Example 1. The sorts ofthe organic solvents used and the amounts of the epoxide formed areshown in Table 8.

                  TABLE 8                                                         ______________________________________                                                        Amounts of the                                                Organic solvents used                                                                         epoxide produced (mg)                                         ______________________________________                                        Paraffins                                                                     n-decane        4.5                                                           n-dodecane      5.1                                                           n-tridecane     5.7                                                           n-tetradecane   6.6                                                           paraffin mixture*                                                                             6.1                                                           Olefins                                                                       1-dodecene      3.5                                                           1-hexadecene    4.7                                                           Halogenated paraffins                                                         1-chlorododecane                                                                              6.0                                                           1-chlorotetradecane                                                                           4.9                                                           Alkylbenzenes                                                                 dodecylbenzene  5.5                                                           tridecylbenzene 5.3                                                           Referential Example                                                           no organic solvent                                                                            2.5                                                           ______________________________________                                         *a mixture of 95% by weight of each normal paraffin of C.sub.9 to C.sub.1     and 5% by weight of each isoparaffin of C.sub.9 to C.sub.16              

EXAMPLE 6

20 ml of a synthetic medium (a liquid medium prepared by addingion-exchanged water to 4 g of (NH₄)₂ HPO₄, 2.5 g of Na₂ HPO₄.12H₂ O, 2 gof KH₂ PO₄, 0.5 g of MgSO₄.7H₂ O, 30 mg of FeSO₄.7H₂ O, 60 mg ofCaCl₂.2H₂ O and 200 mg of yeast extract from "Difco" to make the wholevolume into 1 l and then autoclaved at 120° C. for 15 minutes) placed ina 500 ml shake flask was inoculated with 2 loopfuls of each of the 6microorganisms listed on the following Table 9. The flask was stopperedand 120 ml of propane was introduced into the flask. The flask wasincubated at 30° C. for 120 hours under oscillation. A cell suspensionof each of the 6 microorganisms was prepared according to the methoddescribed in Example 1.

5 ml of the above cell suspension and 250 μl of phenyl allyl ether wereplaced in a test tube having an outer diameter of 24 mm, and the testtube was stoppered. After the incubation at 30° C. for 24 hours underoscillation, the organic layer was extracted with 20 ml of ether and theamount of 2,3-epoxypropyl phenyl ether produced was determined accordingto the method described in Example 1.

The results are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                                                       Amounts                                                              Cell     of the                                                               concen-  epoxide                                                              trations produced                                       Microorganism used    (mg/l)   (mg)                                           ______________________________________                                        Arthrobacter petroleophaous                                                                  ATCC 21494 0.28     0.23                                       Arthrobacter sp.                                                                             ATCC 27778 1.2      0.04                                       Rhodococcus rhodochrous                                                                      ATCC 29670 4.0      0.05                                       Rhodococcus rhodochrous                                                                      ATCC 29672 4.0      0.04                                       Rhodococcus sp.                                                                              ATCC 29673 1.12     0.05                                       Rhodococcus sp.                                                                              ATCC 29674 1.6      0.08                                       ______________________________________                                    

EXAMPLE 7

Ether was removed from the solution in ether of the product obtained bythe reaction using Arthrobacter rubellus ATCC 21495 as described inExample 1. The residue was dissolved in a mixture of 4 ml of isopropanoland 2 ml of isopropylamine and heated at 80° C. for 4 hours in a 20 mlPyrex ample. Then, the reaction mixture was evaporated to dryness.Thereafter, the residue was dissolved in 10 ml of benzene and theorganic layer was extracted twice with 20 ml of 1N-HCl. To the aqueouslayer was added 20 ml of 6N-NaOH and then the resulting mixture wasextracted with 20 ml of benzene. The benzene layer was dried over Na₂SO₄, and evaporated to dryness. The residue was transferred into a 7 mlvial and then, derivatized with 100 μl ofbis(trimethylsilyl)trifluoro-acetamide under heating at 60 ° C. for 15minutes. Then, 100 μl of 1M solution ofN-heptafluorobutyryl-L-prolylchloride in dichloromethane was added tothe reaction mixture. One μl of the final solution was injected into agaschromatograph equipped with a flame ionization detector and a 60 mlong glass capillary column coated with OV225.

The ratio of the peak areas of the two diastereomers was 74.5 (the peakof shorter retention time) vs. 25.5 (the peak of longer retention time)The optical purity of the 2,3-epoxypropyl phenyl ether produced byArthrobacter rubellus ATCC 21495 was calculated to be 49% e.e.

EXAMPLE 8

According to the same procedure as described in Example 1, cellsuspensions of the 9 microorganisms described in the following Table 10were prepared. The cell concentration of each cell suspension wasadjusted to be within the range of 3.5 to 4.0 g/l in dry cell basis.

REACTION AND ANALYSIS OF THE PRODUCT

Twenty ml of the above cell suspension, 400 μl of allyl benzyl ether and8 ml of n-hexadecane were placed in a ml shake flask. After incubationunder oscillation at 30° C. for 24 hours, the 2,3-epoxypropyl benzylether produced was extracted with 40 ml of ether and its amount wasdetermined according to the method described in Example 1.

RESULTS

The sorts of microorganisms used and the amounts of the epoxide producedby each microorganism are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                                                 Amounts                                                                       of the                                                                        epoxide                                                                       produced                                             Microorganism used       (mg)                                                 ______________________________________                                        Micrococcus paraffinolyticus                                                                    ATCC 15589 0.12                                             Arthrobacter petroleophagus                                                                     ATCC 21494 0.66                                             Arthrobacter roseoparaffinus                                                                    ATCC 15584 0.07                                             Arthrobacter sp.  ATCC 27778 0.09                                             Rhodococcus rhodochrous                                                                         ATCC 29675 0.13                                             Rhodococcus rhodochrous                                                                         ATCC 21197 0.45                                             Nocardia corallina                                                                              ATCC 31338 32.3                                             Rhodoccus sp.     ATCC 15108 0.28                                             Brevibacterium butanicum                                                                        ATCC 21196 0.64                                             ______________________________________                                    

EXAMPLE 9

Using Nocardia corallina ATCC 31338, a cell suspension was prepared bythe same procedure as described in Example 1. Five ml of the cellsuspension was placed in a test tube having an outer diameter of 24 mm,and the reaction was carried out at 30° C. for 24 hours underoscillation according to four kinds of methods, i.e., a reaction method(a) wherein 100 μl of allyl benzyl ether was added to the cellsuspension, a reaction method (b) wherein 100 μl of allyl benzyl etherand 5 ml of n-hexadecane were added to the cell suspension, a reactionmethod (c) wherein 250 μl of allyl benzyl ether was added to the cellsuspension and a reaction method (d) wherein 250 μl of allyl benzylether and 5 ml of n-hexadecane were added to the cell suspension. Theanalysis was made according to the same method as described inExample 1. The amount of the phenylmethoxymethyloxirane produced in eachcase is shown in the following Table 11.

                  TABLE 11                                                        ______________________________________                                        Methods  Amounts of the epoxide produced (mg)                                 ______________________________________                                        (a)      2.5                                                                  (b)      11.9                                                                 (c)      4.0                                                                  (d)      13.6                                                                 ______________________________________                                    

EXAMPLE 10

A cell suspension of Nocardia corallina ATCC 31338 was preparedaccording to the procedure described in Example 1. Five ml of the cellsuspension was placed in a test tube having an outer diameter of 24 min.Then, 100 μl of allyl benzyl ether and 5 ml of each of the organicsolvents described in the following Table 12 were added to the cellsuspension, and the reaction was carried out in the same manner asdescribed in Example 1. After the reaction for 24 hours, the analysis ofthe product was made according to the same method as described inExample 1.

The amounts of the epoxide produced are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                                        Amounts of the                                                Organic solvents used                                                                         epoxide produced (mg)                                         ______________________________________                                        Paraffins                                                                     n-decane        10.5                                                          n-dodecane      13.3                                                          n-tridecane     13.9                                                          n-tetradecane   13.5                                                          paraffin mixture*                                                                             12.2                                                          Olefins                                                                       1-dodecene      12.5                                                          1-hexadecene    13.4                                                          Halogenated paraffins                                                         1-chlorododecane                                                                              11.6                                                          1-chlorotetradecane                                                                           12.5                                                          Alkylbenzenes                                                                 dodecylbenzene  10.8                                                          tridecylbenzene 11.2                                                          ______________________________________                                         *a mixture of 95% by weight of each normal paraffin of C.sub.9 to C.sub.1     and 5% by weight of each isoparaffin of C.sub.9 to C.sub.16              

EXAMPLE 11

20 ml of a synthetic medium (a liquid medium prepared by addingion-exchanged water to 4 g of (NH₄)₂ HPO₄, 2.5 g of Na₂ HPO₄.12H₂ O, 2 gof KH₂ PO₄, 0.5 g of MgSO₄.7H₂ O, 30 mg of FeSO₄.7H₂ O, 600 mg ofCaCl₂.2H₂ O and 200 mg of yeast extract from "Difco" to make the wholevolume into 1 l and then autoclaved at 120° C. for 15 minutes) placed ina 500 ml shake flask was inoculated with 2 loopfuls of Nocardiacorallina ATCC 31338, and the flask was stopperred. Then, 120 ml ofpropylene was introduced into the flask and the flask was incubated at30° C. for 96 hours under oscillation. The cells were washed accordingto the manner described in Example 1, to prepare a cell suspension.

Five ml of the above cell suspension, 250 μl of allyl benzyl ether and 5ml of n-hexadecane were placed in a test tube having an outer diameterof 24 mm. The reaction was carried out in the manner described inExample 2, whereby 6.6 mg of phenylmethoxymethyloxirane was obtained.

EXAMPLE 12

From the ether solution of each of the nine sorts of reaction productsdescribed in Example 8, ether was removed. The residue was dissolved ina mixture of 4 ml of isopropanol and 2 ml of isopropylamine and heatedat 80° C. for 4 hours in a 20 ml Pyrex ample. Then, the reaction mixturewas evaporated to dryness. Thereafter, the residue was dissolved in 10ml of benzene and the organic layer was extracted twice with 20 ml of1N-HCl. To the aqueous layer was added 20 ml of 6N-NaOH and then theresulting mixture was extracted with 20 ml of benzene. The benzene layerwas dried over Na₂ SO₄, and evaporated to dryness. The residue wastransferred into a 7 ml vial and then, derivatized with 100 μl ofbis(trimethylsilyl)trifluoro-acetamide under heating at 60° C. for 15minutes. Then, 100 μl of 1M solution ofN-heptafluorobutyryl-L-prolylchloride in dichloromethane was added tothe reaction mixture. One μl of the final solution was injected into agaschromatograph equipped with a flame ionization detector and a 60 mlong glass capillary column coated with OV225. The absoluteconfiguration and optical purity of the phenylmethoxymethyloxiraneproduced by each of the nine microorganisms are shown in the followingTable 13.

                  TABLE 13                                                        ______________________________________                                                               Absolute Optical                                                              configu- purity                                                               ration   of the                                                               of the   epoxide                                       Microorganism used     epoxide  (% e.e.)                                      ______________________________________                                        Micrococcus paraffinolyticus                                                                  ATCC 15589 S        27                                        Arthrobacter petroleophagus                                                                   ATCC 21494 S        25                                        Arthrobacter roseonaraffinus                                                                  ATCC 15584 S        22                                        Arthrobacter sp.                                                                              ATCC 27778 R        46                                        Rhodococcus rhodochrous                                                                       ATCC 29675 R         5                                        Nocardia butanica                                                                             ATCC 21197 R        32                                        Nocardia corallina                                                                            ATCC 31338 S        10                                        Rhodococcus sp. ATCC 15108 S        21                                        Brevibacterium butanicum                                                                      ATCC 21196 S        26                                        ______________________________________                                    

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A process for the preparation of epoxidescomprising:(a) reacting an allyl ether with Nocardia corallina ATCC31338 under aerobic conditions in an aqueous medium to produce thecorresponding 2,3-epoxypropyl ether and (b) isolating the epoxide,wherein said allyl ether is selected from the group consisting of allylphenyl ether, 2-methylphenyl allyl ether, 3-methylphenyl allyl ether,4-methylphenyl allyl ether, 2-allylphenyl allyl ether, 2-allyloxyphenylallyl ether, 2-ethylphenyl allyl ether, 2-(n-propyl)phenyl allyl etherand allyl benzyl ether.
 2. A process for the preparation of epoxides asclaimed in claim 1, wherein the allyl ether is allyl phenyl ether andthe 2,3-epoxypropyl ether produced is optically active.
 3. A process forthe preparation of epoxides as claimed in claim 1, wherein the reactingstep occurs at a temperature of 20° to 50° C. and a pH of 5 to 9 underaerobic conditions.
 4. A process for the preparation of epoxidescomprising:(a) reacting an allyl ether with Nocardia corallina ATCC31338 under aerobic conditions in an aqueous medium containing awater-insoluble organic solvent to produce the corresponding2,3-epoxypropyl ether, and (b) isolating the epoxide, wherein said allylether is selected from the group consisting of allyl phenyl ether,2-methylphenyl allyl ether, 3-methylphenyl allyl ether, 4-methylphenylallyl ether, 2-allylphenyl allyl ether, 2-allyloxyphenyl allyl ether,2-ethylphenyl allyl ether, 2-(n-propyl)phenyl allyl ether, allyl benzylether, α-naphthyl allyl ether, 2-chlorophenylallyl ether and2-chloro-5-methylphenylallyl ether.
 5. A process for the preparation ofepoxides as claimed in claim 4, wherein the allyl ether is selected fromthe group consisting of 2-methylphenyl alkyl ether, 4-methylphenyl allylether, 2-allylphenyl allyl ether, 2-allyloxyphenyl allyl ether, andα-naphthyl allyl ether, and the 2,3-epoxypropyl ether is opticallyactive.
 6. A process for the preparation of epoxides as claimed in claim4, wherein the water-insoluble organic solvent is selected from thegroup consisting of paraffinic hydrocarbons of C₉ to C₁₇, olefinichydrocarbons of C₁₀ to C₁₈, halogenated paraffins of C₉ to C₁₆,alkylbenzenes having one alkyl side chain of C₆ to C₁₅, and mixturesthereof.
 7. A process for the preparation of epoxides as claimed inclaim 4, wherein the water-insoluble organic solvent is present in anamount of 1 to 200 parts by volume per 100 parts by volume of saidmedium.
 8. A process for the preparation of epoxides as claimed in claim7, wherein the water-insoluble organic solvent is present in an amountof 5 to 100 parts by volume per 100 parts by volume of said medium.
 9. Aprocess for the preparation of epoxides as claimed in claim 4, whereinthe reacting step occurs at a temperature of 20° to 50° C. and a pH of 5to 9 under aerobic conditions.
 10. A process for the preparation ofepoxides as claimed in claim 6, wherein the water-insoluble organicsolvent is a paraffinic hydrocarbon of C₁₂ to C₁₆.
 11. A process for thepreparation of epoxides as claimed in claim 10, wherein the paraffinichydrocarbon of C₁₂ to C₁₆ is an isoparaffinic hydrocarbon of C₁₂ to C₁₆.